Methods for reducing toxicity of a chemotherapeutic drug

ABSTRACT

This disclosure relates to methods for improving the therapeutic index of a chemotherapeutic drug in the treatment of patients afflicted with cancer, by reducing chemotherapy-related toxicity to a level that allows the chemotherapeutic drug to be used in humans.

BACKGROUND

Chemotherapy remains a mainstay for systemic therapy for many types ofcancer, including pancreatic cancer and melanoma. Most chemotherapeuticdrugs are only slightly selective to tumor cells, and toxicity tohealthy proliferating cells can be high (Allen TM. (2002) Cancer2:750-763), often requiring dose reduction and even discontinuation oftreatment. In theory, one way to overcome chemotherapy toxicity issuesas well as improve drug efficacy is to target the chemotherapy drug tothe tumor using antibodies that are specific for proteins selectivelyexpressed (or overexpressed) by tumors cells to attract targeted drugsto the tumor. The desired result is altered bio-distribution of thechemotherapy, with more drug going to the tumor and less affectinghealthy tissue. Despite 30 years of research, however, specifictargeting rarely succeeds in the therapeutic context.

Many chemotherapeutic drugs have been approved by regulatory agencies(e.g., the Food and Drug Administration, FDA) for treatment of varioustypes of cancer. However, many more chemotherapeutic drugs have beenrejected, despite efficacy, because the drug is toxic to one or moretissues in the patient, and such toxicity outweighs any benefit.

This disclosure provides methods for reducing toxicity ofchemotherapeutic agents to improve the therapeutic index.

SUMMARY OF THE INVENTION

The risks of irreversible toxicities, such as directchemotherapy-induced hepatotoxicity or potentiation of preexisting liverdisease, continue to exist for many currently available therapeutics. Itis common for potential chemotherapeutic drugs to be abandoned by drugcompanies or rejected by regulatory agencies because the toxicity tonon-target tissues exceeds the therapeutic benefit. There remains a needfor anti-cancer therapeutics with decreased toxicities that canefficiently target tumor cells in order to treat cancer in a patient.Embodiments herein generally relate to compositions and methods thatresult in improved safety for cancer therapies that otherwise haveunacceptably high toxicity in patients.

The instant technology generally relates to methods for decreasingtoxicity, thereby increasing the therapeutic index, of achemotherapeutic drug by combining the drug with a protein carrier andan antibody or other molecule (e.g., aptamer) that targets the resultingcomplex to an aberrant cell (e.g., tumor cell). It is contemplated thatthe methods as described herein will also increase efficacy of the drug,further increasing the therapeutic index. In some embodiments, thetoxicity of the drug is decreased, at least in part, by an increase inuptake of the drug by the aberrant cells.

In particular, the present disclosure relates to compositions fordecreasing toxicity of and/or providing an acceptable therapeutic indexfor a chemotherapy drug, using antibody therapy with nanoparticlescomprising a protein core, such as albumin, or other biocompatible andpreferably human carrier protein and, associated with the surface ofthat core, antibodies, aptamers, or other proteins (e.g. fusion protein)having a region that associates with the carrier protein/protein corewhile retaining the binding function of the antibody, aptamer or otherbinding agents (e.g., protein) to the target ligand on the surface ofthe particle (e.g., the binding region of the antibody, aptamer or otherbinding agent is exposed outside of the particle or is availablenotwithstanding the interaction of the carrier protein binding portion).

Without being limited to any theory, it is believed that this inventionincreases the therapeutic index by rendering the drug less toxic. Thelower toxicity allows more drug to be delivered while maintainingacceptable side effects. It is also contemplated that the drug is moreefficacious, and as such less drug can be used to get the same resultsprovided by previous compositions. This combination allows for anincrease in the therapeutic index by raising the ceiling and loweringthe floor, and results in an acceptable therapeutic index forchemotherapy agents that otherwise are unacceptable for treating humans.Such a combination is surprising and typically not known.

An acceptable therapeutic index is one which indicates a therapeuticeffect that outweighs toxicity. In some embodiments, an acceptabletherapeutic index is one which would lead to continued pursuit of thechemotherapeutic drug, e.g., clinical trials and/or regulatory agencyapproval.

In one aspect is provided a method for providing an acceptabletherapeutic index of a chemotherapeutic drug targeting aberrantmammalian cells, which method comprises:

-   -   a) combining a therapeutically effective amount of the drug with        a biocompatible protein carrier, wherein the drug has an        unacceptable therapeutic index when administered alone;    -   b) forming a complex with the carrier and an effective amount of        an antibody or aptamer which has specificity to an antigen on        the aberrant cells, wherein the antibodies or aptamers populate        the surface of the complex and retain binding specificity, and    -   c) administering the complex to a patient, wherein        administration enhances delivery of the drug to the cells and        reduces one or more side effects of the drug, thereby increasing        the therapeutic index of the drug to provide an acceptable        therapeutic index.

In one aspect is provided a method for providing an acceptabletherapeutic index of a chemotherapeutic drug targeting tumor cells,which method comprises:

-   -   a) combining a therapeutically effective amount of the drug with        an albumin carrier, wherein the drug has an unacceptable        therapeutic index when administered alone;    -   b) forming a complex with the carrier and an effective amount of        antibody or aptamer which has specificity to an antigen on the        tumor cells, wherein the antibodies or aptamers populate the        surface of the complex and retain binding specificity; and    -   c) administering the complex to a patient wherein administration        enhances delivery of the drug to the tumor cells and reduces one        or more side effects of the drug, thereby increasing the        therapeutic index of the drug.

In one embodiment, the complex is less than 1 micron in diameter. In oneembodiment, the complex has a diameter of between 0.1 and 0.9 microns.

In one aspect, this disclosure relates to a method of reducingchemotherapy drug-related toxicity in a patient having cancer, whichmethod comprises treating the patient with a complex comprising atherapeutically effective amount of a chemotherapy drug with an albumincarrier, and an effective amount of antibody or aptamer which hasspecificity to an antigen on the cancer, wherein the antibodies populatethe surface of the complex and retain binding specificity, wherein thechemotherapy drug has an unacceptable therapeutic index whenadministered alone, such that the patient has reduced risk ofchemotherapy drug-related toxicity.

In one aspect, this disclosure relates to a method for providing anacceptable therapeutic index of a chemotherapeutic drug targetingaberrant mammalian cells, which method comprises:

-   -   a) combining a therapeutically effective amount of the drug with        a biocompatible protein carrier, wherein the drug has an        unacceptable therapeutic index when administered alone;    -   b) forming a complex with the carrier and an effective amount of        binding agent having specificity to the aberrant cells, wherein        the binding agent populates the surface of the complex and        retains specificity, and further wherein the binding agent has a        protein carrier-binding portion; and    -   c) administering the complex to a patient, wherein        administration enhances delivery of the drug to the cells and        reduces one or more side effects of the drug, thereby increasing        the therapeutic index of the drug.

In one embodiment, the binding agents are aptamers, antibodies, fusionproteins, or Fc receptors. Preferably, the binding agent includes acarrier protein-binding portion (e.g., albumin-binding portion), e.g. atan end opposite the binding moiety. It is contemplated that surfacecomplexation of the antibody occurs through the carrier protein-bindingportion of the binding agent, which results in all or part of thecarrier protein-binding portion being associated with the protein core,while the binding portions (regions) (e.g., Fa and Fb portions, nucleicacid, etc.) of the binding agent remain outside of the protein core,thereby retaining their target-specific binding capabilities. In apreferred embodiment, the binding agents are antibodies.

In one embodiment, the aberrant mammalian cells are cancer cells, cellsinvolved in an auto-immune disease, cells involved in an inflammatorydisease, virus-infected cells, or bacteria-infected cells.

In one embodiment, the protein carrier is albumin, gelatin, elastin,gliadin, legumin, zein, soy protein, milk protein, or whey protein.Preferably, the protein carrier is albumin.

In one embodiment, the complex comprises an effective amount ofpaclitaxel to provide stability to the complex. In one embodiment, theamount of paclitaxel is less than the therapeutically effective amountof paclitaxel.

In one embodiment, drug-related toxicity is reduced. In one embodiment,the chemotherapy drug-related toxicity is cardiotoxicity,nephrotoxicity, hepatotoxicity, pulmonary toxicity, dermatologictoxicity, or gastrointestinal toxicity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph indicating the average radiant efficiency per unitarea of background or tumors in mice injected with alexaflor 750-labeledABRAXANE, ABRAXANE coated with non-specific antibody (AB IgG) orABRAXANE coated with Rituximab (AR160).

DETAILED DESCRIPTION

After reading this description it will become apparent to one skilled inthe art how to implement the invention in various alternativeembodiments and alternative applications. However, all the variousembodiments of the present invention will not be described herein. Itwill be understood that the embodiments presented here are presented byway of an example only, and not limitation. As such, this detaileddescription of various alternative embodiments should not be construedto limit the scope or breadth of the present invention as set forthbelow.

Before the present invention is disclosed and described, it is to beunderstood that the aspects described below are not limited to specificcompositions, methods of preparing such compositions, or uses thereof assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

The detailed description of the invention is divided into varioussections only for the reader's convenience and disclosure found in anysection may be combined with that in another section. Titles orsubtitles may be used in the specification for the convenience of areader, which are not intended to influence the scope of the presentinvention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In this specification and inthe claims that follow, reference will be made to a number of terms thatshall be defined to have the following meanings.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”. “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

The term “about” when used before a numerical designation. e.g.,temperature, time, amount, concentration, and such other, including arange, indicates approximations which may vary by (+) or (−) 10%, 5%,1%, or any subrange or subvalue there between. Preferably, the term“about” when used with regard to a dose amount means that the dose mayvary by +/−10%.

“Comprising” or “comprises” is intended to mean that the compositionsand methods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination for the stated purpose. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude other materials or steps that do not materially affectthe basic and novel characteristic(s) of the claimed invention.“Consisting of” shall mean excluding more than trace elements of otheringredients and substantial method steps. Embodiments defined by each ofthese transition terms are within the scope of this invention.

The term “antibody” or “antibodies” as used herein refers toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules (i.e., molecules that contain an antigenbinding site that immuno-specifically bind an antigen). The term alsorefers to antibodies comprised of two immunoglobulin heavy chains andtwo immunoglobulin light chains as well as a variety of forms includingfull length antibodies and portions thereof; including, for example, animmunoglobulin molecule, a monoclonal antibody, a chimeric antibody, aCDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, aFv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), adiabody, a multispecific antibody, a dual specific antibody, ananti-idiotypic antibody, a bispecific antibody, a functionally activeepitope-binding fragment thereof, bifunctional hybrid antibodies (e.g.,Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and single chains(e.g., Huston et al.. Proc. Natl. Acad. Sci. USA., 85, 5879-5883 (1988)and Bird et al., Science 242, 423-426 (1988), which are incorporatedherein by reference). (See, generally. Hood et al., Immunology,Benjamin. N.Y., 2ND ed. (1984); Harlow and Lane, Antibodies. ALaboratory Manual, Cold Spring Harbor Laboratory (1988); Hunkapiller andHood, Nature, 323, 15-16 (1986), which are incorporated herein byreference). The antibody may be of any type (e.g., IgG, IgA, IgM, IgE orIgD). Preferably, the antibody is IgG. More preferably, the antibodycontains a Fc domain. An antibody may be non-human (e.g., from mouse,goat, or any other animal), fully human, humanized, or chimeric. Where aparticular antibody (e.g., bevacizumab) is recited herein as theantibody, it is contemplated that a different antibody can besubstituted.

The term “antigen” is well understood in the art and includes substanceswhich are immunogenic. As used herein, the term “antigen” may also referto a substance to which a binding agent other than an antibody (e.g., anaptamer) can bind.

The term “aptamer” as used herein relates to a single-stranded DNA orRNA molecule or peptide that binds to a target, for example, smallmolecules, toxins, peptides, proteins, viruses, bacteria, and even wholecells. Aptamers can be engineered and then selected from large randomsequence pools. To increase stability and binding affinity, nucleic acidaptamers may include unnatural or modified bases and/or a mini hairpinstructure.

The term “binding agent” is generic to antibodies, aptamers modified tocontain a protein carrier-binding region, fusion proteins, and the like.

The term “biosimilar” as used herein refers to a biopharmaceutical whichis deemed to be comparable in quality, safety, and efficacy to areference product marketed by an innovator company.

The term “carrier protein” or “protein carrier” as used herein refers toproteins that function to transport therapeutic agents, antibodies, orboth. Examples of carrier proteins are discussed in more detail below.Where albumin is recited herein as the carrier protein, it iscontemplated that a different carrier protein can be substituted.

The term “dose” and “dosage” refer to an amount of binding agent (e.g.,antibody or aptamer) or chemotherapeutic drug given to a patient in needthereof. The attending clinician will select an appropriate dose fromthe range based on the patient's weight, age, health, stage of cancer,level of circulating antigen, and other relevant factors, all of whichare well within the skill of the art. The term “unit dose” refers to adose of the binding agent or chemotherapeutic drug that is given to thepatient to provide a desired result. In some instances, the unit dose issold in a sub-therapeutic formulation (e.g., 10% of the therapeuticdose). The unit dose may be administered as a single dose or a series ofsubdoses. Additionally, some terms used in this specification are morespecifically defined below.

An “effective amount” intends to indicate the amount of a compound oragent (e.g., a chemotherapeutic drug) administered or delivered to thepatient which is most likely to result in the desired treatment outcome.The amount is empirically determined by the patient's clinicalparameters including, but not limited to the stage of disease, age,gender, histology, and likelihood for recurrence. In addition, the levelof circulating antigen can be used to empirically determine theeffective amount of the chemotherapeutic drug and/or binding agent toadminister to a patient.

The term “express” as applied to an antigen, refers to the amount of theantigen produced by a cancer. In one aspect, the amount is determined bymeasuring the expression level of an antigen of interest (e.g., VEGF)for a given patient population or control population (e.g. populationwithout cancer), determining the median expression level of that antigenfor the population, and comparing the expression level of the sameantigen for a patient to the median expression level for the givenpatient population. For example, if the expression level of an antigenof interest for the patient is determined to be above the medianexpression level of the patient population or the control population,that patient is determined to have high expression of the antigen ofinterest. “Overexpression” of an antigen in a sample collected from apatient refers to an increase (i.e., high) of the antigen in the sample.For example, overexpression can be about 1.5 times, or alternatively,about 2.0 times, or alternatively, about 2.5 times, or alternatively,about 3.0 times, or alternatively, about 5 times, or alternatively,about 10 times, or alternatively about 50 times, or yet furtheralternatively more than about 100 times higher than the expression leveldetected in a control sample collected from a person not having cancer.Alternatively, if the expression level of an antigen of interest for thepatient is determined to be below the median expression level of thepatient population, that patient is determined to have low expression ofthe antigen of interest.

The term “hepatic impairment” refers to any liver damage that reducesliver function. Diseases (e.g. hepatitis) or traumatic injury (e.g.,chemical, drugs, alcohol) are non-limiting examples that may reducenormal liver activities.

The terms “lyophilized,” “lyophilzation” and the like as used hereinrefer to a process by which the material (e.g., nanoparticles) to bedried is first frozen and then the ice or frozen solvent is removed bysublimation in a vacuum environment. An excipient may be included inpre-lyophilized formulations to enhance stability of the lyophilizedproduct upon storage. In some embodiments, the carrier protein,therapeutic agent, binding agent, or any combination thereof islyophilized separately. In other embodiments, the carrier protein,therapeutic agent, binding agent, or any combination thereof is firstcombined and then lyophilized. The lyophilized sample may furthercontain additional excipients.

The term “nanoparticle” as used herein refers to particles with at leastone dimension less than 5 microns. In some embodiments, the nanoparticleis less than 1 micron. For direct administration, the nanoparticle maybe larger. Even larger particles are expressly contemplated by theinvention. The terms “conjugate” and “complex” as used herein aresynonymous with “nanoparticle.” The term “nanoparticle” may alsoencompass discrete multimers of smaller unit nanoparticles. For example,a 320 nm particle comprises a dimer of a unit 160 nm nanoparticle. For160 nm nanoparticles, multimers would therefore be approximately 320 nm,480 nm, 640 nm, 800 nm, 960 nm, 120 nm, and so on as determined by aMastersizer 2000 (available from Malvern Instruments Ltd, Wocestershire,UK) as described in PCT/US15/54295.

In a population of particles, the size of individual particles isdistributed about a mean. Particle sizes for the population cantherefore be represented by an average, and also by percentiles. D50 isthe particle size below which 50% of the particles fall, 10% ofparticles are smaller than the D10 value and 90% of particles aresmaller than D90. Where unclear, the “average” size is equivalent toD50.

As used herein, the term “therapeutic index” with regard to achemotherapeutic drug (agent) indicates safety of the chemotherapeuticdrug. In some aspects, the therapeutic index can include a comparison ofthe amount of a therapeutic agent that causes the therapeutic effect(e.g., killing cancer cells) to the amount of the therapeutic agent thatcauses toxicity (e.g., liver toxicity). The larger the therapeuticindex, the safer the drug is. It is contemplated that according tocertain embodiments an improved therapeutic index can occur using thecompositions and/or methods described herein, including withoutlimitation when: (1) the dosage of chemotherapeutic agent is increasedabove the current therapeutic dosages; (2) the dosage ofchemotherapeutic agent remains the same as the current therapeuticdosages; or (3) the dosage of chemotherapeutic agent is decreased belowthe current therapeutic dosages. In some embodiments, the compositionsand methods, including the specifically numbered scenarios in thisparagraph can elicit improved or similar therapeutic effect as seen withthe current therapeutic dosages with no worse, fewer, or no toxicities.

As used herein, the phrase “unacceptable therapeutic index” refers to atherapeutic index that is too low for the drug to be pursued as achemotherapeutic drug. That is, the toxicity of the drug to a patientoutweighs any therapeutic effect, such that a drug company or clinicalresearcher would not pursue the drug as a potential therapeutic drug(e.g., would not have additional clinical or pre-clinical trials withthe drug), and/or a regulatory agency (e.g., the FDA) would not approvethe drug for use.

As used herein, the term “therapeutic effect” refers to achievement ofthe desired and/or beneficial consequences of a medical treatment. Anon-limiting example of a therapeutic effect of the present disclosureis the shrinkage and/or eradication of a tumor and/or killing of cancercells in a patient.

The term “treating” or “treatment” covers the treatment of a disease ordisorder (e.g., cancer), in a subject, such as a human, and includes:(i) inhibiting a disease or disorder, i.e., arresting its development,(ii) relieving a disease or disorder, i.e. causing regression of thedisorder; (iii) slowing progression of the disorder; and/or (iv)inhibiting, relieving, or slowing progression of one or more symptoms ofthe disease or disorder. In some embodiments “treating” or “treatment”refers to the killing of cancer cells. In some embodiments “treating” or“treatment” refers to increasing progression-free survival of thepatient(s). In some embodiments “treating” or “treatment” refers toincreasing survival rates.

An effective amount or a therapeutically effective amount or dose of anagent, e.g., a compound of the invention, refers to that amount of theagent or compound that results in amelioration of symptoms or aprolongation of survival in a subject. Toxicity and therapeutic efficacyof such molecules can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals. e.g., bydetermining the LD50 (the dose lethal to 50% of the population) and theED50 (the dose therapeutically effective in 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index,which can be expressed as the ratio LD50/ED50. Agents that exhibit hightherapeutic indices are preferred.

A “therapeutically effective amount of paclitaxel” is an amount ofpaclitaxel which is generally used to treat cancer in a patient. Forexample, the recommended dose for adults, depending on the cancer to betreated, is 50 milligrams per square meter of patient surface area(mg/m²) to 175 mg/m². See. e.g., www.drugs.com/dosage/paclitaxel.html.Thus, “less than a therapeutically effective amount” or “sub-therapeuticamount” of paclitaxel refers to an amount of paclitaxel that is lessthan the therapeutic amount, e.g., 0.1 mg/m² to 100 mg/m², or 0.1 mg/m¹to 50 mg/m², or 1 mg/m² to 50 mg/m², or 1 mg/m² to 40 mg/m². The amountmay be or any subrange or value between any ranges provided.

A “stable” formulation is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage. For example, various analyticaltechniques for measuring protein stability are available in the art andare reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent LeeEd., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A.Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at aselected temperature for a selected time period.

Methods

As will be apparent to the skilled artisan upon reading this disclosure,the present disclosure relates to methods for reducing toxicity, therebyimproving the therapeutic index of a chemotherapeutic drug in thetreatment of a patient having aberrant cells. In a preferred embodiment,the patient is afflicted with cancer.

The risks of irreversible toxicities, such as directchemotherapy-induced hepatotoxicity or potentiation of preexisting liverdisease, continue to exist for many currently available therapeutics. Itis common for potential chemotherapeutic drugs to be abandoned by drugcompanies or rejected by regulatory agencies because the toxicity tonon-target tissues exceeds the therapeutic benefit. There remains a needfor anti-cancer therapeutics with decreased toxicities that canefficiently target tumor cells in order to treat cancer in a patient.Embodiments herein generally relate to compositions and methods thatresult in improved safety for cancer therapies that otherwise have anunacceptable therapeutic index.

In particular, the present disclosure relates to methods for increasingthe therapeutic index of a chemotherapeutic drug (e.g., loweringtoxicity, increasing tumor uptake of the drug, increasing efficacy,etc.) by combining the drug with a protein carrier and an antibody orother molecule that targets the resulting complex to an aberrant cell(e.g., tumor cell).

An acceptable therapeutic index is one which indicates a therapeuticeffect that outweighs toxicity. In some embodiments, an acceptabletherapeutic index is one which would lead to continued pursuit of thechemotherapeutic drug, e.g., clinical trials and/or regulatory agencyapproval.

In one aspect is provided a method for providing an acceptabletherapeutic index of a chemotherapeutic drug targeting aberrantmammalian cells, which method comprises:

-   -   a) combining a therapeutically effective amount of the drug with        a biocompatible protein carrier, wherein the drug has an        unacceptable therapeutic index when administered alone;    -   b) forming a complex with the carrier and an effective amount of        an antibody or aptamer which has specificity to an antigen on        the aberrant cells, wherein the antibodies or aptamers populate        the surface of the complex and retain binding specificity; and    -   c) administering the complex to a patient, wherein        administration enhances delivery of the drug to the cells and        reduces one or more side effects of the drug, thereby increasing        the therapeutic index of the drug to provide an acceptable        therapeutic index.

In one aspect is provided a method for providing an acceptabletherapeutic index of a chemotherapeutic drug targeting tumor cells,which method comprises:

-   -   a) combining a therapeutically effective amount of the drug with        an albumin carrier, wherein the drug has an unacceptable        therapeutic index when administered alone;    -   b) forming a complex with the carrier and an effective amount of        antibody or aptamer which has specificity to an antigen on the        tumor cells, wherein the antibodies or aptamers populate the        surface of the complex and retain binding specificity, and    -   c) administering the complex to a patient wherein administration        enhances delivery of the drug to the tumor cells and reduces one        or more side effects of the drug, thereby increasing the        therapeutic index of the drug.

In one embodiment, the complex is less than 1 micron in diameter.

In one aspect, this disclosure relates to a method of reducingchemotherapy drug-related toxicity in a patient having cancer, whichmethod comprises treating the patient with a complex comprising atherapeutically effective amount of a chemotherapy drug with an albumincarrier, and an effective amount of antibody or aptamer which hasspecificity to an antigen on the cancer, wherein the antibodies populatethe surface of the complex and retain binding specificity, wherein thechemotherapy drug has an unacceptable therapeutic index whenadministered alone, such that the patient has reduced risk ofchemotherapy drug-related toxicity.

In one aspect, this disclosure relates to a method for providing anacceptable therapeutic index of a chemotherapeutic drug targetingaberrant mammalian cells, which method comprises:

-   -   a) combining a therapeutically effective amount of the drug with        a biocompatible protein carrier, wherein the drug has an        unacceptable therapeutic index when administered alone;    -   b) forming a complex with the carrier and an effective amount of        binding agent having specificity to the aberrant cells, wherein        the binding agent populates the surface of the complex and        retains specificity, and further wherein the binding agent has a        protein carrier-binding portion; and    -   c) administering the complex to a patient, wherein        administration enhances delivery of the drug to the cells and        reduces one or more side effects of the drug, thereby increasing        the therapeutic index of the drug.

In one embodiment, the binding agents are aptamers, antibodies, fusionproteins, or Fc receptors. Preferably, the binding agent includes aprotein carrier-binding portion, e.g. at an end opposite the bindingmoiety. It is contemplated that surface complexation of the antibodyoccurs through the protein carrier-binding portion (e.g.,albumin-binding portion) of the binding agent (such as the Fc componentof the antibodies recited herein), while the binding portions (regions)(e.g., Fa and Fb portions, nucleic acid, etc.) of the binding agentremain outside of the protein core, thereby retaining theirtarget-specific binding capabilities.

In one embodiment, the aberrant mammalian cells are cancer cells, cellsinvolved in an auto-immune disease, cells involved in an inflammatorydisease, virus-infected cells, or bacteria-infected cells.

In one embodiment, the protein carrier is albumin. gelatin, elastin,gliadin, legumin, zein, soy protein, milk protein, or whey protein. In apreferred embodiment, the protein carrier is albumin. In one embodiment,the albumin is human serum albumin (HSA) In one embodiment, the albuminis recombinant albumin, e.g., recombinant HSA.

In one embodiment, drug-related toxicity is reduced. In one embodiment,the chemotherapy drug-related toxicity is cardiotoxicity,nephrotoxicity, hepatotoxicity, pulmonary toxicity, dermatologictoxicity, or gastrointestinal toxicity.

Therapeutic index is a comparison of the amount of a therapeutic agentthat causes the therapeutic effect (e.g., killing cancer cells) to theamount of the therapeutic agent that causes toxicity (e.g., livertoxicity). Toxicities of current formulations of chemotherapeutic drugsare known include increased hepatic impairment. As the liver is the siteof metabolism for most chemotherapeutic drugs, many agents arehepatotoxic (directly or indirectly). Administration of suchtherapeutics to patients with hepatic impairments is known to includeincreased myelosuppression such that these patients must be monitoredclosely. In addition, some high-risk patients are recommended to notreceive chemotherapeutic drugs at all.

Other known toxicities that may result from chemotherapy treatmentinclude, but are not limited to, cardiotoxicity, nephrotoxicity,pulmonary toxicity, dermatologic toxicity, and gastrointestinaltoxicity. For example, some chemotherapeutic drugs may cause directinjury to the heart (either acute or chronic). Chemotherapy drugsproduce urinary tract/kidney toxicity. Drugs with pulmonary toxicity cancause severe pulmonary effects. Dermatologic toxicity is also commonwith chemotherapeutic drugs, and include transient rash,photosensitivity, dermatitis, hyperpigmentation, urticaria, nailchanges, alopecia, and radiation recall. Gastrointestinal toxicity,including stomatitis or diarrhea, is also common.

In particular, it is contemplated that chemotherapeutic drugs that havea high level of toxicity will benefit from administration ofchemotherapeutic drugs in combination with nanoparticles, as describedherein. That is, it is contemplated that administration of thechemotherapeutic drug as a nanoparticle (complex) as described hereinwill result in decreased toxicity of the drug (e.g., to non-targettissues). It is further contemplated that administration of thechemotherapeutic drug as a nanoparticle will result in increasedefficacy of the drug. Thus, the combination of the chemotherapeutic drugwith the protein core and a targeting antibody may increase thetherapeutic index of the drug by both reducing side effects andimproving efficacy of the drug, and may result in a formulation of thechemotherapeutic drug that has an acceptable therapeutic index and canbe pursued/approved for use in humans having the target disease.

In some embodiments, the patient is screened for hepatic impairment (orrisk thereof) prior to administration of the drug. Determination ofpatients with hepatic impairment or at risk of hepatic impairment can bedetermined by any method known to those of skill in the art.Non-limiting examples of ways to determine severity of hepaticimpairment include The Child-Pugh classification. This classificationsystem groups patients on the basis of two clinical features(encephalopathy and ascites) and three laboratory based parameters(S-albumin. S-bilirubin, and prothrombin time). Increased albumin isdue, at least in part, to decreased synthesis by the hepatocytes inchronic liver disease. Increased levels of bilirubin may be due tocholestasis, hepatocellular failure or extrahepatic causes such ashemolysis. The use of markers like serum albumin. prothrombin time andbilirubin is encouraged and abnormalities in these parameters may bebetter related to drug elimination capacity than other components of theChild-Pugh classification. e.g. encephalopathy and ascites. Impairedhepatic metabolic capacity can also be tested by administration of aprobe drug (e.g., CYP3A4) and observing altered pharmacokinetics of theprobe. Exogenous markers that have been used to assess different hepaticdrug elimination mechanisms are antipyrine, MEGX (lidocaine metabolite),ICG (indocyanine green) and galactose. These, and other, methods can beused alone or in combination to determine whether a patient suffers oris at risk of hepatic impairment.

Paclitaxel has been associated with hepatotoxicity including elevationof serum aminotransferase in approximately 7-26% of patients, withlevels greater than 5 times the upper limit of normal in approximately2% of those receiving paclitaxel. It has been suggested that liverinjury that arises during therapy is due, at least in part, to a directeffect of paclitaxel in inhibiting microtubular function.

It is contemplated in some embodiments that an improved therapeuticindex can occur using the compositions and/or methods described herein,for example, when: (1) the dosage of chemotherapeutic drug is increasedabove the current therapeutic dosages; (2) the dosage ofchemotherapeutic drug remains the same as the current therapeuticdosages; or (3) the dosage of chemotherapeutic drug is decreased belowthe current therapeutic dosages. In some embodiments, the compositionsand methods, including the specifically numbered scenarios in thisparagraph can elicit improved or similar therapeutic effect as seen withthe current therapeutic dosages with no worse, fewer or no toxicities.

In some embodiments, the carrier protein can be albumin, gelatin,elastin (including topoelastin) or elastin-derived polypeptides (e.g.,α-elastin and elastin-like polypeptides (ELPs)), gliadin, legumin, zein,soy protein (e.g., soy protein isolate (SPI)), milk protein (e.g.,$3-lactoglobulin (BLG) and casein), or whey protein (e.g., whey proteinconcentrates (WPC) and whey protein isolates (WPI)). In preferredembodiments, the carrier protein is albumin. In preferred embodiments,the albumin is egg white (ovalbumin), bovine serum albumin (BSA), or thelike. In even more preferred embodiments, the carrier protein is humanserum albumin (HSA). In some embodiments, the carrier protein is agenerally regarded as safe (GRAS) excipient approved by the UnitedStates Food and Drug Administration (FDA).

In some embodiments, the antibody or aptamer targets a non-cell membranebound antigen, for example, VEGF. A commercially available antibody thattargets VEGF is AVASTIN®/bevacirumab and biosimilars thereof in someembodiments, the antibody or aptamer binds to a tumor related antigen, anon-tumor related antigen, or both. A tumor related antigen is anantigenic substance produced in or by tumor cells. It is within theability of one of skill in the art to determine what is a tumor relatedantigen.

Table I depicts a list of non-limiting list of antibodies for cancertargets.

TABLE 1 Antibodies for cancer targets Antibodies BiologicTreatment(s)/Target(s) Monoclonal antibodies Rituximab (RITUXAN ®)Non-Hodgkin lymphoma (MAbs) Alemtuzumab (CAMPATH ®) Chronic lymphocyticleukemia (CLL) Ipilimumab (YERVOY ®) Metastatic melanoma Bevacizumab(AVASTIN ®) Colon cancer, lung cancer, renal cancer, ovarian cancer,glioblastoma multiforme Cetuximab (ERBITUX ®) Colorectal cancer,non-small cell lung cancer, head and neck cancer, cervical cancer,glioblastoma, ovarian epithelia, fallopian tube or primary peritonealcancer, renal cell cancer Panitumumab (VECTIBIX ®) Colorectal cancerTrastuzumab (HERCEPTIN ®) Breast cancer, Adenocarcinoma ⁹⁰Y-ibritumomabtiuxetan Non-Hodgkin lymphoma (ZEVALIN ®) Ado-trastuzumab emtansineBreast cancer (KADYCLA ®, also called TDM-1) Brentuximab vedotin Hodgkinlymphoma, (ADCETRIS ®) Anaplastic large cell lymphoma Blinatumomab(BLINCYTO) Acute lymphocytic leukemia (ALL) Pembrolizumab PD-1(melanoma, non-small (KEYTRUDA ®) cell lung cancer) Nivolumab (OPDIVO ®)PD-1 (melanoma, non-small cell lung cancer) Ofatumumab (ARZERRA ®)Chronic lymphocytic leukemia (CLL) Pertuzumab (PERJETA ®) Breast cancerObinutuzumab (GAZYVA ®) Lymphoma Dinutuximab (UNITUXIN ®) NeuroblastomaDenosumab (PROLIA ®) Bone metastases, multiple myeloma, giant cell tumorof bone

In some embodiments, the antibody is selected from the group consistingof ado-trastuzumab emtansine, alemtuzumab, bevacizumab, blinatumomab,brentuximab vedotin, cetuximab, denosumab, dinutuximab, ibritumomabtiuxetan, ipilimumab, nivolumab, obinutuzumab, ofatumumab, panitumumab,pembrolizumab, pertuzumab, rituximab, trastuzumab, or any biosimilarthereof.

In one embodiment, the antibody or other binding agent comprises aprotein carrier-binding domain. The protein carrier-binding domain maybe any region, domain, amino acid sequence, etc. which allows forinteraction (e.g., hydrophobic interaction) between the protein carrier(e.g., albumin) and the binding agent (or portion thereof). In oneembodiment, the binding agent is covalently bound to the albumin orother carrier protein. In one embodiment, the binding agent is bound tothe albumin or other carrier protein via hydrophobic interactions.

In some aspects, the complexes and compositions as described hereintarget non-cancer diseases. Non-cancer diseases include, withoutlimitation, inflammatory diseases, autoimmune diseases, and infectiousdiseases. In one embodiment, the antibody or other binding agent isspecific for an epitope associated with an infectious disease. In oneembodiment, the disease is caused by a pathogen selected from the groupconsisting of bacteria, fungus, virus, or parasite infection. In oneembodiment, the antibody or other binding agent is specific for anepitope associated with the pathogen. In one embodiment, the antibody orother binding agent is specific for an epitope associated with a toxinproduced by the pathogen. In one embodiment, the antibody or otherbinding agent targets one or more symptoms of the infectious disease.

Tables 2 and 3 depict non-limiting lists of antibodies and fusionproteins for infectious disease targets.

TABLE 2 Antibodies and Fusion Protein for Infectious Disease (approvedor in trials) Antibodies Biologic Type Treatment(s) Target(s)Palivizumab Humanized Respiratory syncytial RSV F antibody virus proteinActoxumab Human antibody Clostridium difficile Exotoxin colitis TcdABezlotoxumab Human antibody Clostridium difficile Exotoxin infectionTcdB N/A Fusion protein: Bacterial sepsis Toll-like receptor 4 with IgG1Fc

TABLE 3 Other antibodies for infectious disease uses Antibodies AntibodyType Proposed Treatment/Target Bezlotoxumab human Clostridium difficilecolitis CR6261 human infectious disease, influenza A Diridavumab humaninfluenza A Edobacomab mouse sepsis caused by Gram-negative bacteriaEfungumab human invasive Candida infection Exbivirumab human hepatitis BFelvizumab humanized respiratory syncytial virus infection Firivumabhuman influenza Foravirumab human Rabies Ibalizumab humanized HIVinfection Libivirumab human hepatitis B Motavizumab humanizedrespiratory syncytial virus Obiltoxaximab chimeric Bacillus anthracisspores Pagibaximab chimeric sepsis (Staphylococcus) Panobacumab humanPseudomonas aeruginosa infection Pritoxaximab chimeric Anti-Shiga toxin1 B subunit PRO 140 humanized HIV infection VRC01LS humanized HIVRafivirumab human Rabies Raxibacumab human anthrax (prophylaxis andtreatment) Regavirumab human cytomegalovirus infection Setoxaximabchimeric E. coli Sevirumab human cytomegalovirus infection Suvizumabhumanized viral infections Tefibazumab humanized Staphylococcus aureusinfection Tosatoxumab human Anti-S. aureus alpha-toxin Tuvirumab humanchronic hepatitis B Urtoxazumab humanized diarrhoea caused by E. coli

In one embodiment, the antibody is specific for an epitope associatedwith a non-cancer disease. In one embodiment, the disease is anautoimmune disease. In one embodiment, the disease is an allergy. In oneembodiment, the disease is asthma. In one embodiment, the disease isassociated with inflammation or an inflammatory response. Preferably,the disease is not an infectious disease.

Tables 4-6 depict non-limiting lists of antibodies or fusion proteinsfor non-oncology targets, e.g., autoimmune disease or inflammatorydisease.

TABLE 4 Antibodies approved or in trials for non-oncology targets, e.g.,autoimmune disease or inflammatory disease. Antibodies Biologic TypeTreatment(s) Target(s) abciximab Chimeric Cardiovascular diseaseinhibition of glycoprotein IIb/IIIa basiliximab Chimeric Transplantrejection CD25 certolizumab Humanized Crohn's disease; RA TNF daclizumabHumanized Transplant rejection CD25 eculizumab Humanized Paroxysmalnocturnal C5 hemoglobinuria complement protein efalizumab HumanizedPsoriasis CD11a infliximab Chimeric Autoimmune disorders TNFmuromonab-CD3 Murine Transplant rejection T-cell CD3 receptornatalizumab Humanized Multiple Sclerosis; Crohn's α4 integrin diseasesubunit omalizumab Humanized Asthma, eczema, allergy IgEtocilizumab/atlizumab Humanized Rheumatoid arthritis (RA); IL-6R JIAvedolizumab Humanized Crohn's disease; ulcerative α4β7 integrin colitisabrilumab Human inflammatory bowel disease; α4β7 integrin ulcerativecolitis; Crohn's disease adalimumab Human RA, JIA, psoriatic arthritis,TNF Crohn's disease, AS and plaque psoriasis belimumab Human Systemiclupus BAFF erythematosus canakinumab Human Cryopyrin-associated IL-1βperiodic syndrome (CAPS), arthritis; gout; neonatal-onset multisysteminflammatory disease golimumab Human Arthritis; Ankylosing TNFspondylitis (AS) ustekinumab Human Psoriatic Arthritis; Plaque IL-12 andIL- Psoriasis; Crohn's disease 23 otelixizumab chimeric/humanizeddiabetes mellitus type 1 CD3 teplizumab humanized diabetes mellitus type1 CD3 ocrelizumab humanized rheumatoid arthritis, lupus CD20erythematosus etc. Alemtuzumab humanized Multiple sclerosis CD52Mepolizumab humanized asthma and white blood cell IL-5 diseases;Hyper-eosinophilic syndrome Reslizumab humanized inflammations of theIL-5 airways, skin and gastrointestinal tract; Eosinophilic oesophagitisranibizumab Humanized Macular degeneration VEGF-A Briakinumab humanpsoriasis, rheumatoid IL-12 and IL- arthritis, inflammatory bowel 23diseases, multiple sclerosis

TABLE 5 Fusion proteins approved or in trials for non-oncology targets,e.g., autoimmune disease or inflammatory disease Fusion ProteinsBiologic Description Treatment(s) Target(s) Aflibercept VEGF receptorWet macular VEGF fragment with IgG1 degeneration; colorectal Fc cancerbelatacept CTLA-4 with IgG1 Organ rejection T cell Fc activationrilonacept IL-1R with IgG1 Fc Cryopyrin-associated IL-1 periodicsyndromes; romiplostim Thrombopoietin- Thrombocytopenia Activationbinding peptide with of TPO IgG1 Fc receptor abatacept Mutated CTLA-4Rheumatoid arthritis CD80 and with IgG1 Fc CD86 alefacept LFA-3 withIgG1 Fc Psoriasis, transplant CD2 rejection etanercept TNFR with IgG1 FcRheumatoid arthritis; TNF juvenile idiopathic arthritis (JIA);psoriasis; ankylosing spondylitis N/A glucagon like Type I diabetespeptide 1 with IgG2 Atacicept TACI ECD-Fc Systemic lupus BAFF and (IgG1)fusion erythematosus; graft APRIL protein, modified Fc vs host diseaseto eliminate effector functions

TABLE 6 Other antibodies for non-oncology uses Antibodies Antibody TypeProposed Treatment/Target Alirocumab human hypercholesterolemiaAnifrolumab human systemic lupus erythematosus Anrukinzumab humanizedUlcerative colitis Aselizumab humanized severely injured patientsAtinumab human Anti-reticulon 4 Atlizumab humanized rheumatoid arthritisAtorolimumab human hemolytic disease of the newborn Begelomab mousegraft versus host disease Benralizumab humanized Asthma Bertilimumabhuman severe allergic disorders Bimagrumab human myostatin inhibitorBimekizumab humanized arthritis Blosozumab humanized osteoporosisBococizumab humanized dyslipidemia Brodalumab human inflammatorydiseases Brolucizumab humanized psoriatic arthritis Caplacizumabhumanized thrombotic thrombocytopenic purpura, thrombosis Cedelizumabhumanized prevention of organ transplant rejections, treatment ofautoimmune diseases Clazakizumab humanized rheumatoid arthritisClenoliximab chimeric rheumatoid arthritis Concizumab humanized bleedingDaptrolizumab humanized Lupus pegol Dectrekumab human allergic rhinitis(hay fever), allergic asthma, rectal fistula in patients with Crohn'sdisease, oesophagitis and pulmonary fibrosis Dupilumab human atopicdiseases Eldelumab human Crohn's disease, ulcerative colitis Elsilimomabmouse immunosuppression Enlimomab mouse Arthritis/transplant rejectionpegol Enokizumab humanized asthma. Etrolizumab humanized inflammatorybowel disease Evinacumab human dyslipidemia Evolocumab humanhypercholesterolemia Fanolesomab mouse appendicitis (diagnosis)Fasinumab human acute sciatic pain Fezakinumab human rheumatoidarthritis, psoriasis Fletikumab human rheumatoid arthritis Fontolizumabhumanized Crohn's disease etc. Foralumab human Inflammatory diseasesFresolimumab human idiopathic pulmonary fibrosis, focal segmentalglomerulosclerosis, cancer Fulranumab human Pain Gavilimomab mouse graftversus host disease Gevokizumab humanized diabetes etc. Gomiliximabchimeric allergic asthma Guselkumab human psoriasis Idarucizumabhumanized reversal of anticoagulant effects of dabigatran Inclacumabhuman inflammation Inolimomab mouse graft versus host disease Itolizumabhumanized psoriasis Ixekizumab humanized autoimmune diseases Keliximabchimeric chronic asthma Lambrolizumab humanized antineoplastic agentLampalizumab humanized Macular degeneration Lebrikizumab humanizedasthma Lerdelimumab human reduction of scarring after glaucoma surgeryLigelizumab humanized severe asthma and chronic spontaneous urticariaLodelcizumab humanized hypercholesterolemia Lulizumab pegol humanizedautoimmune diseases Maslimomab mouse immunosuppression Mavrilimumabhuman rheumatoid arthritis Metelimumab human systemic sclerodermaMorolimumab human Anti-Rhesus factor Namilumab human psoriasis Nebacumabhuman sepsis Nemolizumab humanized Atopic dermatitis Nerelimomab mouseTNF inhibitor Odulimomab mouse prevention of organ transplantrejections, immunological diseases Olokizumab humanized Inflammatorydisease Opicinumab human multiple sclerosis Orticumab human Inflammatorydisease Oxelumab human asthma Ozanezumab humanized ALS and multiplesclerosis Ozoralizumab humanized inflammation Pascolizumab humanizedasthma Pateclizumab humanized TNF Perakizumab humanized arthritisPexelizumab humanized reduction of side effects of cardiac surgeryPlaculumab human Inflammatory diseases Priliximab chimeric Crohn'sdisease, multiple sclerosis Quilizumab humanized asthma Ralpancizumabhumanized dyslipidemia Refanezumab humanized recovery of motor functionafter stroke Rinucumab human neovascular age-related maculardegeneration Roledumab human anti-RhD Romosozumab humanized osteoporosisRontalizumab humanized systemic lupus erythematosus Rovelizomabhumanized haemorrhagic shock etc. Ruplizumab humanized rheumaticdiseases Sarilumab human rheumatoid arthritis, ankylosing spondylitisSecukinumab human uveitis, rheumatoid arthritis psoriasis Sifalimumabhumanized SLE, dermatomyositis, polymyositis Simtuzumab humanizedfibrosis Siplizumab humanized psoriasis, graft-versus-host disease(prevention) Sirukumab human rheumatoid arthritis Sonepcizumab humanizedchoroidal and retinal neovascularization Sontuzumab humanizednon-alcoholic steatohepatitis/primary sclerosing cholangitis Stamulumabhuman muscular dystrophy Tadocizumab humanized percutaneous coronaryintervention Talizumab humanized allergic reaction Tanezumab humanizedpain Telimomab mouse Immunosuppressive (linked to A chain aritox ofricin protein) Teneliximab chimeric Anti-CD40 Tesidolumab humanChoroiditis; Dry age-related macular degeneration; Panuveitis;Paroxysmal nocturnal haemoglobinuria; Wet age- related maculardegeneration TGN1412 humanized chronic lymphocytic leukemia, rheumatoidarthritis Tildrakizumab humanized immunologically mediated inflammatorydisorders Toralizumab humanized rheumatoid arthritis, lupus nephritisetc. Tralokinumab human asthma etc. Tregalizumab humanized Anti-CD4Trevogrumab human muscle atrophy due to orthopedic disuse and sarcopeniaVatelizumab humanized Multiple sclerosis Vepalimomab mouse inflammationVisilizumab humanized Crohn's disease, ulcerative colitis Zanolimumabhuman rheumatoid arthritis, psoriasis, T-cell lymphoma Zolimomab mousesystemic lupus erythematosus, graft- aritox versus-host disease

In some aspects, the nanoparticle composition further comprises atherapeutic agent. In one embodiment, the therapeutic agent is anantibiotic or antimicrobial. In one embodiment, the therapeutic agent isan anti-inflammatory agent. Such therapeutic agents are known in theart. In some aspects, the complex further comprises a sub-therapeuticamount of paclitaxel, which amount is sufficient to allow formation ofthe complex.

Aptamers are DNA or RNA molecules that can bind to a target molecule(e.g., a protein expressed by the cancer cell or aberrant cell). Thisdisclosure employs aptamers that target aberrant cells, such as cancercells or virus-infected cells. Aptamers are selected based on theirrelative binding affinities to the molecule of interest from a libraryof nucleic acids or peptides. The library can be as large as 1015members—preferably either single strand DNA or RNA. Methodology toisolate aptamers having strong binding affinities is reported byDeGrasse, PLoS One, 2012, 7(3) e33410, which is incorporated herein byreference in its entirety.

Like an antibody, an aptamer can specifically bind to its target withpicomolar to nanomolar affinity. Importantly, unlike antibodies,aptamers can be directly amplified by PCR. Aptamers have been widelyused in many applications, including target detection, enzymeinhibition, receptor regulation, and drug delivery. Some aptamers (e.g.,MACUGEN, for age-related macular degeneration) have been approved by theFDA, and several show promise towards various diseases, includingcancer. See, e.g., Wu et al., Theranostics. 2015; 5(4): 322-344, whichis incorporated herein by reference in its entirety.

Without being bound by theory, it is contemplated that the combinationof unglycosylated or partially glycosylated (e.g., as compared to thenaturally-occurring or human-derived) antibody or fusion protein mayalter its binding capability to a protein core. In such cases, where thecarrier-binding portion is present in a region of the antibody or fusionprotein that is unglycosylated or partially glycosylated, the proteinwill coat or bind to the portion of an aptamer or fusion protein thatinteracts with the protein core (e.g., albumin), thereby reducing theimmunogenicity of the binding agent while imparting increased stabilityand/or efficacy of the antibody, the aptamer or fusion protein in vivo.

In some embodiments, the antibody is a non-therapeutic andnon-endogenous human antibody. In some embodiments, the antibody is achimeric antibody, a non-endogenous human antibody, a humanizedantibody, or non-human antibody.

In some embodiments, the chemotherapeutic drug (agent) is selected fromthe group consisting of abiraterone, bendamustine, bortezomib,carboplatin, cabazitaxel, cisplatin, chlorambucil, dasatinib, docetaxel,doxorubicin, epirubicin, erlotinib, etoposide, everolimus, gefitinib,idarubicin, imatinib, hydroxyurea, imatinib, lapatinib, leuprorelin,melphalan, methotrexate, mitoxantrone, nedaplatin, nilotinib,oxaliplatin, paclitaxel, pazopanib, pemetrexed, picoplatin, romidepsin,satraplatin, sorafenib, vemurafenib, sunitinib, teniposide, triplatin,vinblastine, vinorelbine, vincristine, and cyclophosphamide.

Both ABRAXANE® and albumin particles comprising other chemotherapeuticagents are disclosed by U.S. Pat. Nos. 7,758,891; 7,820,788; 7,923,536;8,034,375; 8,138,229; 8,268,348; 8,314,156; 8,853,260; and 9,101,543,each of which is incorporated herein by reference in its entirety. Inaddition, carrier protein, chemotherapeutic drug, antibody conjugates,or combinations thereof are disclosed by PCT/US2015/054295 and U.S.Publication No. 2014/0178486, each of which is incorporated herein byreference in its entirety.

In some embodiments, the chemotherapeutic agent is associated with acarrier protein. In some embodiments, the complex further comprises asub-therapeutic amount of paclitaxel.

In some embodiments, the effective amount of the chemotherapeutic drugis selected from an amount consisting of about 100 mg/m², about 105mg/m², about 110) mg/m², about 115 mg/m², about 120 mg/m², about 125mg/m², about 130 mg/m², about 135 mg/m², about 140 mg/m², about 145mg/m², about 150 mg/m², about 155 mg/m², about 160 mg/m² about 165mg/m², about 170 mg/m², about 175 mg/m², about 180 mg/m², about 185mg/m², about 190 mg/m², about 195 mg/m², or about 200 mg/m² of thechemotherapeutic.

It is to be understood that the therapeutic agent (i.e.,chemotherapeutic agent) may be located inside the nanoparticle, on theoutside surface of the nanoparticle, or both. The nanoparticle maycontain more than one different therapeutic agents, for example, twotherapeutic agents, three therapeutic agents, four therapeutic agents,five therapeutic agents, or more. Furthermore, a nanoparticle maycontain the same or different therapeutic agents inside and outside thenanoparticle.

In one aspect, the amount of chemotherapeutic agent, e.g. paclitaxel, inthe nanoparticle is sufficient to allow formation of the nanoparticle.The use of sub-therapeutic amounts of paclitaxel for formation ofantibody-albumin nanoparticle complexes is described, for example, inU.S. Provisional App. No. 62/384,119, which is incorporated herein byreference in its entirety.

In one embodiment, the amount of paclitaxel present in the nanoparticlecomposition is greater than or equal to a minimum amount capable ofproviding stability to the nanoparticles. In one embodiment, the amountof paclitaxel present in the nanoparticle composition is greater than orequal to a minimum amount capable of providing affinity of the at leastone therapeutic agent to the protein carrier. In one embodiment, theamount of paclitaxel present in the nanoparticle composition is greaterthan or equal to a minimum amount capable of facilitatingcomplex-formation of the at least one therapeutic agent and the proteincarrier. In one embodiment, the weight ratio of the carrier protein andthe paclitaxel of the nanoparticle composition is greater than about9:1. In one embodiment, the weight ratio is greater than about 10:1, or11:1, or 12:1, or 13:1, or 14:1, or 15:1, or about 16:1, or about 17:1,or about 18:1, or about 19:1, or about 20:1, or about 21:1, or about22:1, or about 23:1, or about 24:1, or about 25:1, or about 26:1, orabout 27-1, or about 28:1, or about 29:1, or about 30:1. In oneembodiment, the amount of paclitaxel is equal to an minimum amountcapable of providing stability to the nanoparticles. In one embodiment,the amount of paclitaxel is greater than or equal to a minimum amountcapable of providing affinity of the at least one therapeutic agent tothe protein carrier. In one embodiment, the amount of paclitaxel isgreater than or equal to a minimum amount capable of facilitatingcomplex-formation of the at least one therapeutic agent and the proteincarrier. In any of the embodiments, the amount of paclitaxel can be lessthan a therapeutic amount for paclitaxel. In other words, the amount canbe less than what is provided or contemplated for providing atherapeutic benefit, such as for example, a chemotherapeutic amount toeffectively treat a cancer.

In one embodiment, the amount of paclitaxel present in the nanoparticlecomposition is less than about 5 mg/mL upon reconstitution with anaqueous solution. In one embodiment, the amount of paclitaxel present inthe nanoparticle composition is less than about 4.54 mg/mL, or about4.16 mg/mL, or about 3.57 mg/mL, or about 3.33 mg/mL, or about 3.12mg/mL, or about 2.94 mg/mL, or about 2.78 mg/mL, or about 2.63 mg/mL, orabout 2.5 mg/mL, or about 2.38 mg/mL, or about 2.27 mg/mL, or about 2.17mg/mL, or about 2.08 mg/mL, or about 2 mg/mL, or about 1.92 mg/mL, orabout 1.85 mg/mL, or about 1.78 mg/mL, or about 1.72 mg/mL, or about1.67 mg/mL upon reconstitution with an aqueous solution.

In some embodiments any antibody, aptamer, therapeutic agent, or anycombination thereof is expressly excluded.

Cancers or tumors that can be treated by the compositions and methodsdescribed herein include, but are not limited to cancers listed in theabove tables and: biliary tract cancer; brain cancer, includingglioblastomas and medulloblastomas; breast cancer; uterine cancer; tubalcancer; cervical cancer; choriocarcinoma; colon cancer; bladder cancer;endometrial cancer; vaginal cancer; vulvar cancer; esophageal cancer;mouth cancer; gastric cancer; kidney cancer; hematological neoplasms,including acute lymphocytic and myelogenous leukemia; multiple myeloma;AIDS associated leukemias and adult T-cell leukemia lymphoma;intraepithelial neoplasms, including Bowen's disease and Paget'sdisease; liver cancer (hepatocarcinoma); lung cancer; head or neckcancers or oral cancers (mouth, throat, esophageal, nasopharyngeal, jaw,tonsil, nasal, lip, salivary gland, tongue, etc.); lymphomas, includingHodgkin's disease and lymphocytic lymphomas; neuroblastomas;neuroendocrine tumors; oral cancer, including squamous cell carcinoma;adrenal cancer, anal cancer; angiosarcoma; appendix cancer; bile ductcancer; bone cancer; carcinoid tumors; soft tissue sarcoma;rhabdomyosarcoma; eye cancer; ovarian cancer, including those arisingfrom epithelial cells, stromal cells, germ cells and mesenchymal cells,and fallopian tube cancer; gallbladder cancer, pancreas cancer; prostatecancer; rectal cancer; sarcomas, including leiomyosarcoma,rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma; skincancer, including melanoma, Kaposi's sarcoma, basocellular cancer andsquamous cell cancer; testicular cancer, including germinal tumors(seminoma, non-seminoma[teratomas, choriocarcinoma]), stromal tumors andgerm cell tumors; penile cancer; hemangioendothelioma; gastrointestinalcancer, ureteral cancer; urethral cancer; spinal cancer; pituitary glandcancer; primary central nervous system (CNS) lymphoma; thyroid cancer,including thyroid adenocarcinoma and medullar carcinoma; and renalcancer including adenocarcinoma and Wilms tumor. In importantembodiments, cancers or tumors include breast cancer, prostate cancer,colorectal cancer, lymphoma, multiple myeloma, and melanoma

In some cases, complexes as described herein can be designed to have anaverage diameter that is less than 1 μm. For example, appropriateconcentrations of carrier protein and antibody (or other binding agent)can be used such that complexes having an average diameter that is lessthan 1 μm are formed. In some cases, the complexes provided herein canhave an average diameter that is between 0.1 μm and 1 μm (e.g., between0.1 μm and 0.95 μm, between 0.1 μm and 0.9 μm, between 0.1 μm and 0.8μm, between 0.1 μm and 0.7 μm, between 0.1 μm and 0.6 μm, between 0.1 μmand 0.5 μm, between 0.1 μm and 0.4 μm, between 0.1 μm and 0.3 μm,between 0.1 μm and 0.2 μm, between 0.2 μm and 1 μm, between 0.3 μm and 1μm, between 0.4 μm and 1 μm, between 0.5 μm and 1 μm, between 0.2 μm and0.6 μm, between 0.3 μm and 0.6 μm, between 0.2 μm and 0.5 μm, or between0.3 μm and 0.5 μm). Complexes provided herein having an average diameterthat is between 0.1 μm and 0.9 μm can be administered systemically(e.g., intravenously) to treat cancer or other disease located within amammal's body.

In some cases, a complex as provided herein can have greater than 60percent (e.g., greater than 65, 70, 75, 80, 90, 95, or 99 percent) ofthe complexes having a diameter that is between 0.1 μm and 0.9 μm (e.g.,between 0.1 μm and 0.95 μm, between 0.1 μm and 0.9 μm, between 0.1 μmand 0.8 μm, between 0.1 μm and 0.7 μm, between 0.1 μm and 0.6 μm,between 0.1 μm and 0.5 μm, between 0.1 μm and 0.4 μm, between 0.1 μm and0.3 μm, between 0.1 μm and 0.2 μm, between 0.2 μm and 1 μm, between 0.3μm and 1 μm, between 0.4 μm and 1 μm, between 0.5 μm and 1 μm, between0.2 μm and 0.6 μm, between 0.3 μm and 0.6 μm, between 0.2 μm and 0.5 μm,or between 0.3 μm and 0.5 μm). Complexes provided herein having greaterthan 60 percent (e.g., greater than 65, 70, 75, 80, 90, 95, or 99percent) of the complexes with a diameter that is between 0.1 μm and 0.9μm can be administered systemically (e.g., intravenously) to treatcancer or other disease expressing the relevant antigen located within amammal's body.

In general, any appropriate combination of carrier protein, chemotherapyagent, and binding agent can be used as described herein. For example,an appropriate amount of carrier protein (e.g., with a chemotherapeuticdrug), and an appropriate amount of binding agent can be mixed togetherin the same container. This mixture can be incubated at an appropriatetemperature (e.g., room temperature, between 5° C. and 60° C., between23° C. and 60° C., between 15° C. and 30° C., between 15° C. and 25° C.,between 20° C. and 30° C., or between 20° C. and 25° C.) for a period oftime (e.g., about 30 minutes, or between about 5 minutes and about 60minutes, between about 5 minutes and about 45 minutes, between about 15minutes and about 60 minutes, between about 15 minutes and about 45minutes, between about 20 minutes and about 400 minutes, or betweenabout 25 minutes and about 35 minutes) before being administered to apatient having a cancer.

In some cases, carrier protein nanoparticles comprising a chemotherapyagent can be contacted with a binding agent to form complexes that arestored prior to being administered to a patient. For example, acomposition can be formed as described herein and stored for a period oftime (e.g., days or weeks) prior to being administered to a patient.

Any appropriate method can be used to obtain complexes as describedherein. Any appropriate method can be used to administer a complex asprovided herein to a mammal. For example, a composition containingcarrier protein/binding agent/chemotherapeutic drug complexes can beadministered via injection (e.g., subcutaneous injection, intramuscularinjection, intravenous injection, or intrathecal injection).

Before administering a composition containing a complex as providedherein to a mammal, the mammal can be assessed to determine whether ornot the mammal has a cancer or disease expressing the relevant antigen.Any appropriate method can be used to determine whether or not a mammalhas a cancer or disease expressing the relevant antigen. For example, amammal (e.g., human) can be identified using standard diagnostictechniques. In some cases, a tissue biopsy can be collected and analyzedto determine whether or not a mammal has a cancer or disease expressingthe antigen.

After identifying a mammal as having the disease or cancer, the mammalcan be administered a composition containing a complex as providedherein. For example, a composition containing the complex can beadministered prior to or in lieu of surgical resection of a tumor. Insome cases, a composition containing a complex as provided herein can beadministered following resection of a tumor.

In some cases the nanoparticle complex as described herein may beadministered with an effective amount of NK or NK-92 cells. The NK orNK-92 cells may be administered to the subject concurrently with thecomplexes or may be administered sequentially to the subject. Forexample, the NK-92 cells may be administered before the complexes areadministered to the subject. An effective amount of the NK or NK-92cells can be any amount that further reduces the progression rate of acancer or disease expressing the antigen recognized by the binding agent(e.g., antibody or aptamer), increases the progression-free survivalrate, or increases the median time to progression as compared using thecomplexes without the NK or NK-92 cells, and preferably withoutproducing significant toxicity to the mammal.

If a particular mammal fails to respond to a particular amount, then theamount can be increased by, for example, two fold. After receiving thishigher concentration, the mammal can be monitored for bothresponsiveness to the treatment and toxicity symptoms, and adjustmentsmade accordingly. The effective amount can remain constant or can beadjusted as a sliding scale or variable dose depending on the mammal'sresponse to treatment. Various factors can influence the actualeffective amount used for a particular application. For example, thefrequency of administration, duration of treatment, use of multipletreatment agents, route of administration, and severity of the cancer ordisease may require an increase or decrease in the actual effectiveamount administered.

A composition containing a complex as provided herein can beadministered to a mammal in any appropriate amount, at any appropriatefrequency, and for any appropriate duration effective to achieve adesired outcome (e.g., to increase progression-free survival). In somecases, a composition as provided herein can be administered to a mammalhaving a cancer or disease to reduce the progression rate of the canceror disease by 5, 10, 25, 50, 75, 100, or more percent. For example, theprogression rate can be reduced such that no additional cancerprogression is detected.

Any appropriate method can be used to determine whether or not theprogression rate of cancer is reduced. For example, the progression rateof a cancer can be assessed by imaging tissue at different time pointsand determining the amount of cancer cells present. The amounts ofcancer cells determined within tissue at different times can be comparedto determine the progression rate. After treatment as described herein,the progression rate can be determined again over another time interval.In some cases, the stage of cancer after treatment can be determined andcompared to the stage before treatment to determine whether or not theprogression rate was reduced.

In some cases, a composition as provided herein can be administered to amammal having a cancer under conditions where progression-free survivalis increased (e.g., by 5, 10, 25, 50, 75, 100, or more percent) ascompared to the median progression-free survival of correspondingmammals having untreated cancer or the median progression-free survivalof corresponding mammals having cancer treated with the carrier protein,chemotherapy agent, and the binding agent without forming complexesprior to administration. In some cases, a composition as provided hereincan be administered to a mammal having a cancer to increaseprogression-free survival by 5, 10, 25, 50, 75, 100, or more percent ascompared to the median progression-free survival of correspondingmammals having a cancer and having received the carrier protein,chemotherapy agent, carrier protein/chemotherapy agent nanoparticle(without a binding agent), or binding agent alone. Progression-freesurvival can be measured over any length of time (e.g., one month, twomonths, three months, four months, five months, six months, or longer).

In some cases, a composition containing a complex as provided herein canbe administered to a mammal having a under conditions where the 8-weekprogression-free survival rate for a population of mammals is 65% orgreater (e.g., 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80% or greater) than that observed in a population ofcomparable mammals not receiving a composition containing complexes asprovided herein. In some cases, the composition can be administered to amammal having a cancer under conditions where the median time toprogression for a population of mammals is at least 150 days (e.g., atleast 155, 160, 163, 165, or 170 days).

An effective amount of a composition containing complexes as providedherein can be any amount that reduces the progression rate of a canceror disease expressing the antigen recognized by the binding agent,increases the progression-free survival rate, or increases the mediantime to progression without producing significant toxicity to themammal. If a particular mammal fails to respond to a particular amount,then the amount can be increased by, for example, two fold. Afterreceiving this higher concentration, the mammal can be monitored forboth responsiveness to the treatment and toxicity symptoms, andadjustments made accordingly. The effective amount can remain constantor can be adjusted as a sliding scale or variable dose depending on themammal's response to treatment. Various factors can influence the actualeffective amount used for a particular application. For example, thefrequency of administration, duration of treatment, use of multipletreatment agents, route of administration, and severity of the cancer ordisease may require an increase or decrease in the actual effectiveamount administered.

The frequency of administration can be any frequency that reduces theprogression rate of a cancer or disease, increases the progression-freesurvival rate, or increases the median time to progression withoutproducing significant toxicity to the mammal. For example, the frequencyof administration can be from about once a month to about three times amonth, or from about twice a month to about six times a month, or fromabout once every two months to about three times every two months. Thefrequency of administration can remain constant or can be variableduring the duration of treatment. A course of treatment with acomposition as provided herein can include rest periods. For example,the composition can be administered over a two week period followed by atwo week rest period, and such a regimen can be repeated multiple times.As with the effective amount, various factors can influence the actualfrequency of administration used for a particular application. Forexample, the effective amount, duration of treatment, use of multipletreatment agents, route of administration, and severity of the cancer ordisease may require an increase or decrease in administration frequency.

An effective duration for administering a composition provided hereincan be any duration that reduces the progression rate of a cancer ordisease, increases the progression-free survival rate, or increases themedian time to progression without producing significant toxicity to themammal. Thus, the effective duration can vary from several days toseveral weeks, months, or years. In general, the effective duration forthe treatment of a cancer or disease can range in duration from severalweeks to several months. In some cases, an effective duration can be foras long as an individual mammal is alive. Multiple factors can influencethe actual effective duration used for a particular treatment. Forexample, an effective duration can vary with the frequency ofadministration, effective amount, use of multiple treatment agents,route of administration, and severity of the cancer or disease.

A composition containing carrier protein/chemotherapy agent/bindingagent complexes as provided herein can be in any appropriate form. Forexample, a composition provided herein can be in the form of a solutionor powder with or without a diluent to make an injectable suspension. Acomposition also can contain additional ingredients including, withoutlimitation, pharmaceutically acceptable vehicles. A pharmaceuticallyacceptable vehicle can be, for example, saline, water, lactic acid,mannitol, or combinations thereof.

After administering a composition provided herein to a mammal, themammal can be monitored to determine whether or not the cancer ordisease was treated. For example, a mammal can be assessed aftertreatment to determine whether or not the progression rate of the canceror disease was reduced (e.g., stopped). As described herein, any methodcan be used to assess progression and survival rates.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention Other aspects, advantages, and modifications are within thescope of the following claims.

EXAMPLES

One skilled in the art would understand that descriptions of making andusing the particles described herein is for the sole purpose ofillustration, and that the present disclosure is not limited by thisillustration.

Example 1. Albumin Nanoparticles Comprising Rituximab (AR160)

The particles are synthesized by adding between about 5 mg and about 20mg of rituximab (or non-specific IgG) to 20 mg of ABRAXANE. Saline isthen added to a final volume of 2 ml for a final concentration of 10mg/ml ABRAXANE, and the mixture is allowed to incubate at roomtemperature for 30 minutes to allow particle formation. Particlesaverage about 160 nm and are termed “AR160” nanoparticles.

Optionally, the composition is divided into aliquots and frozen at −80°C. Once frozen the aliquots are optionally lyophilized overnight withthe Virtis 3L benchtop lyophilizer (SP Scientific, Warmister, Pa.) withthe refrigeration on. A lyophilized preparation is generated.

The dried aliquots are stored at room temperature. These samples arereconstituted in saline at room temperature for 30 minutes, followed bycentrifugation for 7 minutes at 2000×g. The resulting sample is thenresuspended in the appropriate buffer, as needed

Example 2. Evaluation of Tumor Uptake of AR160

Mice were injected via subcutaneous injection with lymphoma cells andtumors allowed to form. Mice received intravenous (IV) injection ofequal amounts of alexaflor 750-labeled ABRAXANE (ABX), ABRAXANE coatedwith non-specific antibodies (AB IgG), or AR160.

Twenty-four hours after IV injection, tumor accumulation of therespective treatments was determined based on a fluorescence threshold.Background was determined based on a region of the mouse without a tumorFIG. 1 is a graphical representation of background and tumorfluorescence. Table 8 indicates the numerical values for each, includingtumor-associated fluorescence (average radiant efficiency from the tumorminus background). Addition of rituximab to the ABRAXANE nanoparticle(AR160) results in a nearly 100% increase in tumor uptake of ABRAXANE.

TABLE 8 Average Radiant Efficiency and Adjusted Tumor-AssociatedFluorescence Tumor- associated Background Tumor Fluorescence ABX 1.5412.09 0.549 AB IgG 1.4005 1.99 0.5895 AR160 1.545 2.637 1.092

1-11. (canceled)
 12. A method of reducing chemotherapy drug-relatedtoxicity in a patient having cancer, which method comprises treatingsaid patient with a complex comprising a therapeutically effectiveamount of a chemotherapy drug with an albumin carrier, and an effectiveamount of antibody which has specificity to an antigen on said cancer,wherein said antibodies populate the surface of said complex and retainbinding specificity, wherein the chemotherapy drug has an unacceptabletherapeutic index when administered alone, such that said patient hasreduced risk of chemotherapy drug-related toxicity.
 13. The method ofclaim 12, wherein the chemotherapy drug-related toxicity is selectedfrom the group consisting of cardiotoxicity, nephrotoxicity,hepatotoxicity, pulmonary toxicity, dermatologic toxicity, andgastrointestinal toxicity. 14-20. (canceled)
 21. The method of claim 12,wherein the complex further comprises an effective amount of paclitaxelto form said complex.
 22. The method of claim 21, where the amount ofpaclitaxel is a sub-therapeutic amount.
 23. The method of claim 21,wherein the amount of paclitaxel is between 0.1 mg/m² and 50 mg/m². 24.The method of claim 21, wherein the weight ratio of the albumin carrierand the paclitaxel of the complex is greater than about 9:1.
 25. Themethod of claim 12, wherein the average diameter of the complex is lessthan 1 micron.
 26. The method of claim 12, wherein the antibody isselected from the group consisting of ado-trastuzumab emtansine,alemtuzumab, bevacizumab, blinatumomab, brentuximab vedotin, cetuximab,denosumab, dinutuximab, ibritumomab tiuxetan, ipilimumab, nivolumab,obinutuzumab, ofatumumab, panitumumab, pembrolizumab, pertuzumab,rituximab, trastuzumab, any combination thereof, and any biosimilarthereof.
 27. The method of claim 12, wherein the antibody is ananti-CD20 antibody which has specificity to a CD20 antigen.
 28. Themethod of claim 27, wherein the antibody is rituximab.
 29. The method ofclaim 12, wherein the complex further comprises a therapeutic agent. 30.The method of claim 29, wherein the therapeutic agent is an antibiotic,an antimicrobial, an anti-inflammatory agent, or any combinationthereof.
 31. The method of claim 12, wherein the chemotherapy drug isselected from the group consisting of abiraterone, bendamustine,bortezomib, carboplatin, cabazitaxel, cisplatin, chlorambucil,dasatinib, docetaxel, doxorubicin, epirubicin, erlotinib, etoposide,everolimus, gefitinib, idarubicin, imatinib, hydroxyurea, imatinib,lapatinib, leuprorelin, melphalan, methotrexate, mitoxantrone,nedaplatin, nilotinib, oxaliplatin, paclitaxel, pazopanib, pemetrexed,picoplatin, romidepsin, satraplatin, sorafenib, vemurafenib, sunitinib,teniposide, triplatin, vinblastine, vinorelbine, vincristine,cyclophosphamide, and any combination thereof.
 32. The method of claim12, wherein the albumin carrier comprises ovalbumin, bovine serumalbumin, human serum albumin, or any combination thereof.
 33. The methodof claim 12, wherein the average diameter of the complex is between 0.1μm and 0.9 μm.
 34. The method of claim 12, wherein at least 60 percentof complexes have a diameter between 0.1 μm and 0.9 μm.
 35. The methodof claim 12, wherein the complex is administered intravenously.
 36. Themethod of claim 12, wherein the complex is administered with aneffective amount of NK or NK-92 cells.
 37. The method of claim 36,wherein the NK or NK-92 cells are administered concurrently with thecomplex, sequentially to the complex, or a combination thereof.
 38. Themethod of claim 12, wherein a progression rate of the cancer is reducedby at least 5, at least 10, at least 25, at least 50, at least 75, or atleast 100 percent.